SUMMARY Kinetoplastid parasites are single-celled eukaryotic parasites, some of which are causative agents of devastating human diseases, including Chagas disease, Leishmaniasis, and human African trypanosomiasis. Pathogenic kinetoplastids are transmitted by insect vectors. These parasite-vector relationships are specific, with different insect species harboring different species of parasite. While the life cycles of kinetoplastid parasites in their respective insect hosts can differ, one shared feature is adherence of the parasite to insect tissue. The adhesive stage is necessary for colonization of the insect, and in some cases allows for development of infectious forms. For all kinetoplastids, the adhesion itself has shared ultrastructural features, and resembles a hemidesmosome. The molecular components of this adhesive structure, and the signaling pathways that trigger its formation, are completely unknown. Crithidia fasciculata is a parasite that only infects one host, the mosquito. It is generally not considered to be a human pathogen; however, there have been reports of human infections, typically in immunocompromised patients or in co-infections with Leishmania spp. C. fasciculata has for years been used as a model for exploring the basic biology of kinetoplastid parasites. They represent a powerful system to investigate mechanisms of adhesion since they will adhere not only to the hindgut of their mosquito host, but to artificial substrates such as tissue culture plastic. This allows us to use in vitro assays to determine the role of various candidate proteins and pathways in the process of adhesion. In addition, we can observe the stages of the adhesion process in real time using live-cell imaging. We hypothesize that the adhesive stage of the parasite is a distinct developmental form, and that differentiation to this form is mediated by specific signal transduction pathways. In addition, we predict that adhesion is a multi-stage process involving novel proteins. We will address these hypotheses through the following Specific Aims: (1) Determine the role of the cyclic AMP signaling pathway in regulating adhesion, and (2) Establish conditions for rapid creation of genetic knock-outs in C. fasciculata using CRISPR/Cas9. This project builds upon our published work using RNAseq to compare gene expression profiles of adherent and swimming cells, and will set the stage for a high-throughput approach to determine the role of a large number of candidate proteins in adhesion in vitro, which can then be evaluated in vivo for their ability to colonize mosquitoes. The outcomes of the proposed work will be improved tools for genetic manipulation of C. fasciculata, which will benefit researchers using this model, and insight into shared mechanisms for adhesion of diverse kinetoplastid species to their insect hosts.